BGM1004/L06 Recombinant DNA Technology II Flashcards
Why don’t all plasmids contain the desired DNA insert?
Some relegate back to their original form as they are all cut by the same enzyme
What gene is disrupted by insertion of DNA fragments into polylinker?
lacZ (beta-galactosidase)
What colour do bacteria containing the pUC18 insert produce?
Blue
Give 2 ways of checking if bacteria contain the desired insert.
Hybridisation to ssDNA probe
PCR using specific primers
Screen for expression
Attach probe to sequence
How does CRISPR-CAS9 guarantee that offspring will inherit the desired trait?
Animal has 2 identical chromosomes
How does CRISPR-CAS9 give 2 identical chromosomes? (2)
Guide RNA directs CAS9 to cut wildtype DNA
Replaced by altered gene in existing chromosomes
Give 3 uses for recombinant DNA.
Induce expression in host
Recover and manipulate further
Recover and transfer to different cell type
Why are proteins directly expressed after DNA recombination? (2)
For purification
To investigate function
Why are modified versions of a protein expressed? (2)
To change properties
To investigate fine detail
What do artificial inducers (IPTG) do?
Stimulate transcription
What do artificial inducers bind to?
Lacl repressor protein
What does a Lacl repressor do?
Prevent expression of cloned gene
How can we ensure that the inserts are oriented correctly?
Use two different restriction enzymes
What are introns?
Non-coding DNA
When are introns removed?
Maturation of mRNA
Why can certain proteins not be made in bacterial cells?
Splicing does not occur in bacterial cells
What occurs as a result of genomic DNA ligation into an expression vector?
Inactive protein expression
What kind of DNA contains the coding sequence with no introns?
cDNA
Give a typical bacterial expression system.
E. coli
B. subtilis
Give 2 advantages to bacterial expression systems.
Simple cells
Short generation times
Large product yield
Low cost
Give 2 disadvantages to bacterial expression systems.
Eukaryotic proteins can fail to fold correctly
Proteins can be toxic to cell
No post-translational modifications
Give 2 advantages to yeast expression systems.
Simple unicellular eukaryote
Resembles mammalian cells
Quick and cheap
Post-translational modifications
Give a disadvantage to yeast expression systems.
Contains proteases
Post-translational modifications may differ from mammalian cells
Give 2 advantages to insect cell expression systems.
Active - high-level expression
Correct folding of proteins
Post-translational modifications
Cheaper than mammalian cell culture
Give a disadvantage to insect cell expression systems.
Post-translational modifications may differ from mammalian cells
Where is insulin made?
Beta-cells in Islets of Langerhans (pancreas)
Give 2 disadvantages to bovine/porcine insulin for diabetes treatment.
Side effects
Difficult to purify
Possible contamination
Describe the structure of insulin.
2 polypeptide chains linked by disulfide bonds
RNA transcribed into chain A&B then joined
What is unfolded insulin called?
Proinsulin
What is removed from proinsulin to form insulin?
Connecting peptide
How many introns does the proinsulin gene contain?
2
How are introns removed when producing recombinant human insulin?
mRNA is reverse-transcribed to produce proinsulin cDNA
What antibodies block HIV infection in vitro?
Anti-CD4
How was the hypothesis that CD4 is the cellular receptor for HIV tested? What was the outcome?
Expressing recombinant CD4 in cells which don’t usually express CD4
These cells became infected
